32 research outputs found
SNARE-mediated membrane fusion trajectories derived from force-clamp experiments.
No full textFusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomic-force microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed
effectors
No full textLegionella pneumophila, the Gram-negative pathogen causing Legionnaires' disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host's disassembly machinery, thus effectively trapping VAMP4 in an inactive state
Author response
No full textThe Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (Ptdlns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to Ptdlns(4,5)P-2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P-2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion
Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs
No full textAbstractThe large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m−1, at which the number of fusion events is increased substantially.</jats:p
« The Desert and the Sown. Imperial Supremacy and Local Culture in Partho-Roman Mesopotamia ». Parthica, 7, 2005, pp. 235-246.
No full textThis paper deals with some aspects of the institutional history of Partho-Roman Mesopotamia, with a restricted focus on the city of Hatra, rightly pointed out as “a strategic key-stone” in the wide context of the Eastern Jezirah. In his further reading of well-known epigraphic evidence, the Author discusses the political environment of this particular area of the Parthian kingdom and the chronology of the city’s sovereigns, recalling their titles in the inscriptions which qualify them either ..
Identifikation und Charakterisierung von synaptischen Proteinen von Drosophila melanogaster mit Hilfe von monoklonalen Antikörpern der Würzburger Hybridoma-Bibliothek
No full textFor a large fraction of the proteins expressed in the human brain only the primary structure is known from the genome project. Proteins conserved in evolution can be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and characterized with the aim to identify the target antigen. The mAb ab52 was found to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15 (epidermal growth factor receptor pathway substrate clone 15) to be a strong candidate. Another mAb from the library, aa2, was already found to recognize EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and 2D electrophoretic separations revealed similar patterns, hence indicating that both antigens could represent the same protein. Finally absence of the wild-type signal in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52 antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for applications like immunoprecipitation (IP). It has already been submitted to the Developmental Studies Hybridoma Bank (DSHB) to be easily available for the entire research community. The mAb na21 was also found to be an IgM. It recognizes a membrane associated antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to biochemically purify the endogenously expressed protein from the tissue, gave promising results but could not be completed due to lack of time. Thus biochemical purification of the protein seems possible in order to facilitate its identification by mass spectrometry. Several other mAbs were studied for their staining pattern on cryosections and whole mounts of Drosophila brains. However, many of these mAbs stained very few structures in the brain, which indicated that only a very limited amount of protein would be available as starting material. Because these antibodies did not produce signals on Western blots, which made it impossible to enrich the antigens by electrophoretic methods, we did not attempt their purification. However, the specific localization of these proteins makes them highly interesting and calls for their further characterization, as they may play a highly specialized role in the development and/or function of the neural circuits they are present in. The purification and identification of such low expression proteins would need novel methods of enrichment of the stained structures.Für einen Großteil der Proteine, die im menschlichen Gehirn exprimiert werden, ist lediglich die Primärstruktur aus dem Genomprojekt bekannt. Proteine, die in der Evolution konserviert wurden, können in genetischen Modellsystemen wie Drosophila untersucht werden. In dieser Doktorarbeit werden monoklonale Antikörper (mAk) aus der Würzburger Hybridoma Bibliothek produziert und charakterisiert, mit dem Ziel, die erkannten Proteine zu identifizieren. Der mAk ab52 wurde als IgM typisiert, das auf Western Blots ein zytosolisches Protein von Mr ~110 kDa erkennt. Das Antigen wurde durch zwei-dimensionale Gelelektrophorese (2DE) als einzelner Fleck aufgelöst. Massenspektrometrische Analyse dieses Flecks identifizierte dass EPS-15 (epidermal growth factor receptor pathway substrate clone 15) als viel versprechenden Kandidaten. Da für einen anderen mAk aus der Bibliothek, aa2, bereits bekannt war, dass er EPS-15 erkennt, wurden die Western-Blot-Signale der beiden Antikörper nach 1D und 2D Trennungen von Kopfhomogenat verglichen. Die Ähnlichkeit der beiden Muster deuteten darauf hin, dass beide Antigene dasselbe Protein erkennen. Das Fehlen des Wildtyp-Signals in homozygoten Eps15 Mutanten in einem Western Blot mit mAk ab52 bestätigten schließlich, dass EPS-15 das Antigen zu mAk ab52 darstellt. Demnach erkennen beide mAk, aa2 und ab52, das Drosophila Homolog zu EPS-15. Da mAk aa2 ein IgG ist, dürfte er für Anwendungen wie Immunpräzipitation (IP) besser geeignet sein. Er wurde daher bereits bei der Developmental Studies Hybridoma Bank (DSHB) eingereicht, um ihn der ganzen Forschergemeinde leicht zugänglich zu machen. Der mAk na21 wurde ebenfalls als IgM typisiert. Er erkennt ein Membran assoziiertes Antigen von Mr ~10 kDa auf Western Blots. Aufgrund der Membranassoziierung des Proteins war es nicht möglich, es in 2DE aufzulösen und da es sich um ein IgM handelt, war eine Anreicherung des Antigens mittels IP nicht erfolgreich. Vorversuche zur biochemischen Reinigung des endogenen Proteins aus Gewebe waren Erfolg versprechend, konnten aber aus Zeitmangel nicht abgeschlossen werden. Daher erscheint eine biochemische Reinigung des Proteins für eine Identifikation durch Massenspektrometrie möglich. Eine Reihe weiterer mAk wurden hinsichtlich ihrer Färbemuster auf Gefrierschnitten und in Ganzpräparaten von Drosophila Gehirnen untersucht. Allerdings färbten viele dieser mAk sehr wenige Strukturen im Gehirn, so dass nur eine sehr begrenzte Menge an Protein als Startmaterial verfügbar wäre. Da diese Antikörper keine Signale auf Western Blots produzierten und daher eine Anreicherung des Antigens durch elektrophoretische Methoden ausschlossen, wurde keine Reinigung versucht. Andererseits macht die spezifische Lokalisation dieser Proteine sie hoch interessant für eine weitere Charakterisierung, da sie eine besonders spezialisierte Rolle in der Entwicklung oder für die Funktion von neuralen Schaltkreisen, in denen sie vorkommen, spielen könnten. Die Reinigung und Identifikation solcher Proteine mit niedrigem Expressionsniveau würde neue Methoden der Anreicherung der gefärbten Strukturen erfordern
Identifikation und Charakterisierung von synaptischen Proteinen von Drosophila melanogaster mit Hilfe von monoklonalen Antikörpern der Würzburger Hybridoma-Bibliothek
No full textFor a large fraction of the proteins expressed in the human brain only the primary
structure is known from the genome project. Proteins conserved in evolution can
be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal
antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and
characterized with the aim to identify the target antigen. The mAb ab52 was found
to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western
blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a
single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15
(epidermal growth factor receptor pathway substrate clone 15) to be a strong
candidate. Another mAb from the library, aa2, was already found to recognize
EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and
2D electrophoretic separations revealed similar patterns, hence indicating that both
antigens could represent the same protein. Finally absence of the wild-type signal
in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52
antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila
homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for
applications like immunoprecipitation (IP). It has already been submitted to the
Developmental Studies Hybridoma Bank (DSHB) to be easily available for the
entire research community.
The mAb na21 was also found to be an IgM. It recognizes a membrane associated
antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature
of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of
the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to
biochemically purify the endogenously expressed protein from the tissue, gave
99
promising results but could not be completed due to lack of time. Thus
biochemical purification of the protein seems possible in order to facilitate its
identification by mass spectrometry. Several other mAbs were studied for their
staining pattern on cryosections and whole mounts of Drosophila brains. However,
many of these mAbs stained very few structures in the brain, which indicated that
only a very limited amount of protein would be available as starting material.
Because these antibodies did not produce signals on Western blots, which made it
impossible to enrich the antigens by electrophoretic methods, we did not attempt
their purification. However, the specific localization of these proteins makes them
highly interesting and calls for their further characterization, as they may play a
highly specialized role in the development and/or function of the neural circuits
they are present in. The purification and identification of such low expression
proteins would need novel methods of enrichment of the stained structures.Für einen Großteil der Proteine, die im menschlichen Gehirn exprimiert werden, ist
lediglich die Primärstruktur aus dem Genomprojekt bekannt. Proteine, die in der
Evolution konserviert wurden, können in genetischen Modellsystemen wie
Drosophila untersucht werden. In dieser Doktorarbeit werden monoklonale
Antikörper (mAk) aus der Würzburger Hybridoma Bibliothek produziert und
charakterisiert, mit dem Ziel, die erkannten Proteine zu identifizieren. Der mAk
ab52 wurde als IgM typisiert, das auf Western Blots ein zytosolisches Protein von
Mr ~110 kDa erkennt. Das Antigen wurde durch zwei-dimensionale
Gelelektrophorese (2DE) als einzelner Fleck aufgelöst. Massenspektrometrische
Analyse dieses Flecks identifizierte dass EPS-15 (epidermal growth factor receptor
pathway substrate clone 15) als viel versprechenden Kandidaten. Da für einen
anderen mAk aus der Bibliothek, aa2, bereits bekannt war, dass er EPS-15 erkennt,
wurden die Western-Blot-Signale der beiden Antikörper nach 1D und 2D
Trennungen von Kopfhomogenat verglichen. Die Ähnlichkeit der beiden Muster
deuteten darauf hin, dass beide Antigene dasselbe Protein erkennen. Das Fehlen
des Wildtyp-Signals in homozygoten Eps15 Mutanten in einem Western Blot mit
mAk ab52 bestätigten schließlich, dass EPS-15 das Antigen zu mAk ab52 darstellt.
Demnach erkennen beide mAk, aa2 und ab52, das Drosophila Homolog zu EPS-
15. Da mAk aa2 ein IgG ist, dürfte er für Anwendungen wie Immunpräzipitation
(IP) besser geeignet sein. Er wurde daher bereits bei der Developmental Studies
Hybridoma Bank (DSHB) eingereicht, um ihn der ganzen Forschergemeinde leicht
zugänglich zu machen.
Der mAk na21 wurde ebenfalls als IgM typisiert. Er erkennt ein Membran
assoziiertes Antigen von Mr ~10 kDa auf Western Blots. Aufgrund der
Membranassoziierung des Proteins war es nicht möglich, es in 2DE aufzulösen und
101
da es sich um ein IgM handelt, war eine Anreicherung des Antigens mittels IP
nicht erfolgreich. Vorversuche zur biochemischen Reinigung des endogenen
Proteins aus Gewebe waren Erfolg versprechend, konnten aber aus Zeitmangel
nicht abgeschlossen werden. Daher erscheint eine biochemische Reinigung des
Proteins für eine Identifikation durch Massenspektrometrie möglich.
Eine Reihe weiterer mAk wurden hinsichtlich ihrer Färbemuster auf
Gefrierschnitten und in Ganzpräparaten von Drosophila Gehirnen untersucht.
Allerdings färbten viele dieser mAk sehr wenige Strukturen im Gehirn, so dass nur
eine sehr begrenzte Menge an Protein als Startmaterial verfügbar wäre. Da diese
Antikörper keine Signale auf Western Blots produzierten und daher eine
Anreicherung des Antigens durch elektrophoretische Methoden ausschlossen,
wurde keine Reinigung versucht. Andererseits macht die spezifische Lokalisation
dieser Proteine sie hoch interessant für eine weitere Charakterisierung, da sie eine
besonders spezialisierte Rolle in der Entwicklung oder für die Funktion von
neuralen Schaltkreisen, in denen sie vorkommen, spielen könnten. Die Reinigung
und Identifikation solcher Proteine mit niedrigem Expressionsniveau würde neue
Methoden der Anreicherung der gefärbten Strukturen erfordern
Application of Python-OpenCV to detect contour of shapes and colour of a real image
No full textAbstract: OpenCV-Python has found its use in various fields during the course of its development, especially in the shape and colour detection of an actual image. It is more reliable to make a better and faster decision. So, OpenCV and Python are increasingly finding their application in the shape and colour detection of the images. In this following work, we will focus on how to detect the shapes and colour of a binary image by using Python 3.8, OpenCV 4.5, and Numpy 1.19.3 resources. This research is investigated in two steps process. Firstly, the image has been processed to detect the various shapes and colors of an image's specific shape. Secondly, catching the colour of an actual image by using our model.
Keywords: OpenCV, Python, Numpy, Computer vision, shape and colour detection.
Title: Application of Python-OpenCV to detect contour of shapes and colour of a real image
Author: Mohammed Khalid Hossen, Sayed Mashequl Bari, Partho Protim Barman, Rana Roy, Pranajit Kumar Das
International Journal of Novel Research in Computer Science and Software Engineering
ISSN 2394-7314
Vol. 9, Issue 2, May 2022 - August 2022
Page No: 20-25
Novelty Journals
Website: www.noveltyjournals.com
Published Date: 24-May-2022
DOI: https://doi.org/10.5281/zenodo.6576264
Paper Download Link (Publisher Website):
https://www.noveltyjournals.com/upload/paper/Application%20of%20Python-24052022-3.pdfInternational Journal of Novel Research in Computer Science and Software Engineering, ISSN 2394-7314, Novelty Journals (Publisher), Website: www.noveltyjournals.co
Adaptive molecular evolution of virulence genes of avian influenza - A virus subtype H5N1: An analysis of host radiation
No full textAdaptive molecular evolution of virulence genes of avian influenza - A virus subtype H5N1: An analysis of host radiation-2
No full text<p><b>Copyright information:</b></p><p>Taken from "Adaptive molecular evolution of virulence genes of avian influenza - A virus subtype H5N1: An analysis of host radiation"</p><p></p><p>Bioinformation 2006;1(8):321-326.</p><p>Published online 26 Dec 2006</p><p>PMCID:PMC1891714.</p><p></p
